Summary Existential challenges to all organisms result from DNA damaging agents present naturally in the environment, e.g., UV radiation and oxygen, and from toxic industrial chemicals. The induction of ?hypermutation?, while perhaps counterintuitive, is essential to counter exposure to environmental stress by ensuring cell and organismic fitness. Hypermutations, mutations occurring at frequencies ~ 10-2 ? 10-3 per base pair, straddle a range between death and fitness in bacteria and humans. The key to fitness is to carefully regulate hypermutation. Our grant proposal is to elucidate the regulation of two essential hypermutator enzymes, DNA polymerase V mutasome (pol V Mut) in Escherichia coli that catalyzes translesion DNA synthesis on damaged DNA templates, and activation-induced deoxycytidine deaminase (AID) required for a robust immune response in humans. Pol V Mut has a multisubunit structure that includes a RecA molecule, the E. coli recombinase, and a molecule of ATP. Along with its polymerase activity, pol V Mut also has an intrinsic DNA-dependent ATPase activity different from all other ATPases. Pol V Mut exists in two conformationally distinct states, activated and deactivated depending on the location of RecA. We hypothesize that the internal ATPase provides an energy source to switch between conformation states, akin to an ?on-off? toggle switch. We propose to test this hypothesis using TIRF-FRET microscopy to visualize the dynamics of switching between each conformational state of pol V Mut at single-molecule resolution, and to use Cryo-EM to determine the location of each pol V subunit, most importantly RecA, in activated and deactivated forms. AID plays an essential role in the immune response by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) in B-cells by deaminating C?U during transcription of immunoglobulin variable (IgV) and switch (IgS) region DNA. We propose to reconstitute the first biochemical system to investigate AID targeting and catalysis during IgV and IgS transcription by human RNA polymerase II. This study is intended to establish a biochemical basis for the hypermutation reactions required in the generation of antibody (Ab) diversity. We propose to use TIRF-FRET microscopy to visualize the action of AID during IgV and IgS transcription, including the influence of proteins believed to be involved in targeting AID to stalled transcription bubbles. In 2016, we obtained a crystal structure for AID. We now propose a strategy to obtain an AID-ssDNA co-crystal structure. Environmental allergens can cause asthma. We will use the co-crystal structure to design AID inhibitors that suppress IgE production to treat asthma. A novel high-risk high-reward project, designed to achieve affinity maturation in a test tube, using AID and error-prone human DNA polymerase ?, is aimed at generating monoclonal Abs against any antigen. As a proof of principal, we propose to generate Abs against three critical ion channel receptors involved in pain, heat and cold. The availability of ion channel receptor Abs would provide a major medical breakthrough to relieve pain and itching in hypersensitive individuals.